Autoactivation of mycorrhizal symbiosis signaling through gibberellin deactivation in orchid seed germination

Abstract

Orchids parasitically depend on external nutrients from mycorrhizal fungi for seed germination. Previous findings suggest that orchids utilize a genetic system of mutualistic arbuscular mycorrhizal (AM) symbiosis, in which the plant hormone gibberellin (GA) negatively affects fungal colonization and development, to establish parasitic symbiosis. Although GA generally promotes seed germination in photosynthetic plants, previous studies have reported low sensitivity of GA in seed germination of mycoheterotrophic orchids where mycorrhizal symbiosis occurs concurrently. To elucidate the connecting mechanisms of orchid seed germination and mycorrhizal symbiosis at the molecular level, we investigated the effect of GA on a hyacinth orchid (Bletilla striata) seed germination and mycorrhizal symbiosis using asymbiotic and symbiotic germination methods. Additionally, we compared the transcriptome profiles between asymbiotically and symbiotically germinated seeds. Exogenous GA negatively affected seed germination and fungal colonization, and endogenous bioactive GA was actively converted to the inactive form during seed germination. Transcriptome analysis showed that B. striata shared many of the induced genes between asymbiotically and symbiotically germinated seeds, including GA metabolism- and signaling-related genes and AM-specific marker homologs. Our study suggests that orchids have evolved in a manner that they do not use bioactive GA as a positive regulator of seed germination and instead autoactivate the mycorrhizal symbiosis pathway through GA inactivation to accept the fungal partner immediately during seed germination.

Figure 1.

Effects of GA (GA₃) and its inhibitor (uniconazole-P) on SPs. A) Symbiotically germinated B. striata seeds at 2 wk after sowing (WAS) on OMA medium inoculated with Tulasnella sp. HR1-1. The images show fungal-infected protocorms stained with calcofluor white (blue) and wheat germ agglutinin-Alexa fluor-488 (green) to visualize the plant cell and fungal structures, respectively. Green fluorescent dots at the suspensor side (arrowheads) and the inset indicate fungal pelotons. Arrows indicate the suspensor end. B, C) The number of symbiotic cells B) and germination percentages C) at 2WAS on OMA medium inoculated with Tulasnella sp. HR1-1. Symbiotic cells and germinated seeds treated with 0.01% (v/v) ethanol for the control, 1 µM GA₃, or 1 µM uniconazole-P were counted. Different letters in B) indicate significant differences among treatments on the basis of a Bonferroni-adjusted pairwise t-test (n = 5 individual experiments, each containing 5 protocorms, P < 0.05). P-value was calculated on the basis of Student's t-test (n = 4 to 5 replicate plates, each containing 185 ± 64 seeds, P < 0.05) C). Each bar represents the mean value ± Sd. All experiments were independently performed 3 times with similar results.

Figure 2.

Effects of GA (GA₃) and its inhibitor (uniconazole-P) on APs. B. striata seeds were sown asymbiotically on Hyponex agar medium A, B) or filter papers C). Germinated seeds treated with 0.01% (v/v) ethanol for the control, GA₃, or uniconazole-P at the indicated concentrations were counted at 2 A, B) or 3 C) wk after sowing. Different letters indicate statistically significant differences on the basis of a Bonferroni-adjusted pairwise t-test (n = 3 to 5 replicate plates, each containing 117 ± 54 seeds, P < 0.05). Each bar represents the mean value ± Sd. All experiments were independently performed 3 times with similar results.

Figure 3.

Transcriptome analysis of asymbiotically and symbiotically germinated B. striata. A) The bar chart of the number of DEGs. Gene expression levels of APs (asymbiotic) and SPs (symbiotic) at 1 to 3 wk after seeding were compared with Week 0 seeds. B) Hierarchical clustering of DEGs. The method of k-means clustering was used to identify similarities in expression patterns among asymbiotic and symbiotic. The heatmap was drawn by the pheatmap package in R. C) GO enrichment analysis of shared overexpressed genes between asymbiotic and symbiotic at Week 1. The most significant 10 terms of each category, biological process, cellular component, and molecular function, are shown on the basis of the elim-Kolmogorov–Smirnov (elimKS) method in the topGO package in R (elimKS < 0.01). All significant terms were presented in Supplemental Table S4.

Figure 4.

The expression patterns of GA signaling and SL biosynthesis genes identified during RNA-seq analyses. A) The expression patterns of SLENDER RICE1 (SLR1) putative orthologs containing DELLA domain. B) The expression patterns of carotenoid cleavage dioxygenase genes (CCD7 and CCD8) involved in SL biosynthesis. The heatmap on the left shows the expression patterns of the selected genes on the basis of Log₂FC. Log₂FC was calculated between time points; 0-wk seeds versus 1- to 3-wk protocorms. “Germination” and “AM symbiosis” indicate germinated seeds and arbuscular mycorrhizal roots of O. sativa, respectively. The right panel displays FDRs.

Figure 5.

Expression of GA biosynthesis and metabolism genes during asymbiotic and symbiotic germination. A) The expression patterns of GA biosynthesis and metabolism genes from RNA-seq data. The heatmap on the left shows the expression patterns of the selected GA-related genes on the basis of Log₂FC. Log₂FC was calculated between time points; 0-wk seeds versus 1 to 3-wk protocorms. “Germination” and “AM symbiosis” indicate germinated seeds and arbuscular mycorrhizal roots of rice, respectively. The right panel displays FDRs. B, C) RT-qPCR of GA biosynthesis and metabolism genes. Relative expression analysis was performed with total RNA isolated from asymbiotically B) or symbiotically C) germinated B. striata at different time points (Week 0 seeds to protocorms at 1 or 2 wk after sowing [WAS]). The relative expression values were determined using the relative standard curve method. The FC in expression is relative to 0-wk-old seeds (expression level = 1). Data are shown as box plots with the heavy line in the box representing the median (50th percentile), the ends of the box representing 25th and 75th percentiles, respectively, and the whiskers showing the smallest and largest value. Black dots and triangles indicate the individual data points. The y-axis scale is logarithmic. Asterisks indicate significant differences compared with the 0-wk-old seeds using a Bonferroni-adjusted pairwise t-test (n = 5 to 9, *P < 0.05).

Figure 6.

Quantification of endogenous GAs in B. striata protocorms. A) Simplified metabolic scheme of GA biosynthesis. The “20ox,” “3ox,” and “2ox” mean GA 20-, 3-, and 2-oxidase enzymes, respectively. B) The content of endogenous GAs during seed germination. The GAs were detected in seeds at Week 0 (“seed”), APs and SPs at Week 2 (“asymbiotic” and “symbiotic,” respectively). Different letters indicate statistically significant differences on the basis of the Bonferroni-adjusted pairwise t-test (n = 3, P < 0.05). Each bar represents the mean value ± Se.

Figure 7.

The expression patterns of symbiosis marker genes of rice and B. striata from RNA-seq data. The heatmap on the left indicates the expression patterns of the arbuscular mycorrhizal symbiosis marker genes (Gutjahr et al. 2008) and the mycorrhiza-specific phosphate transporter PT11 gene on the basis of Log₂FC. In rice, Log₂FC was calculated between Day 0 seeds and germinated seeds at 2 d after seeding (“germination”) and between noncolonized roots and AM roots (“AM symbiosis”). In B. striata, Log₂FC was computed for Week 0 seeds versus 1- to 3-wk protocorms. The right panel displays FDRs. Asterisks indicate the OM symbiosis marker genes (Miura et al. 2018).

Figure 8.

RT-qPCR of OM symbiosis marker genes. Relative expression analysis was performed with total RNA isolated at different time points (Week 0 seeds to germinated protocorms at 2 wk after seeding [WAS]). The relative expression values were determined using the relative standard curve method. Data are shown as box plots with the heavy line in the box representing the median (50th percentile), the ends of the box representing 25th and 75th percentiles, respectively, and the whiskers showing the smallest and largest value. Black and gray dots indicate the individual data points and outliers, respectively. Asterisks indicate significant differences compared with the 0-wk-old seeds using the Bonferroni-adjusted pairwise t-test (n = 4, *P < 0.05).

Figure 9.

Proposed model for seed germination mechanism in orchids. GA stimulates seed germination of AM plants, such as O. sativa, by inducing the expression of α-amylases necessary for the utilization of starch stored in the endosperm (Kaneko et al. 2002). After root development, the mutual relationships between plants and AM fungi are established in the roots. Exogenous-treated GA inhibits fungal colonization in rice through the degradation of DELLA proteins. In B. striata, an OM plant, exogenous treatment with GA inhibits seed germination and fungal colonization via unknown mechanisms. When seed germination occurs, environmental factors probably derived from OM fungi stimulate the expression of GA metabolic genes such as GA3ox and GA2ox, leading to symbiotic signaling even without fungi. OM fungi form pelotons in the cortical layer, which promotes GA2ox gene expression.